More than 25 years ago it was proposed that epimutation—mitotically stable gene silencing associated with epigenetic alteration in DNA methylation—can act as one of Knudson's two hits required for tumorigenesis (1). In subsequent decades many promoter CpG island (CGI) associated genes have been shown to be aberrantly hypermethylated and silenced in various cancers (2). Indeed, recent epigenomic studies revealed that nearly all tumor types harbor hundreds of abnormally hypermethylated promoter CGIs (3), indicating that epimutations are as common in tumors as genetic mutations. Aberrant promoter CGI methylation is associated with distinct environmental exposures (4), gene mutation patterns (5), cancer prognosis (6) and response to therapy (7). Despite the undisputed importance of DNA methylation in cancer, however, its fundamental role in carcinogenesis remains unclear (8-9). Most importantly, it remains unknown whether aberrant DNA methylation is a cause of tumorigenesis (8).
Cancer-related promoter CGI hypermethylation originates in normal tissues, suggesting aberrant methylation could predispose to malignancy (10). p16INK4A (referred to hereafter as p16) is a tumor suppressor gene that regulates the ability of retinoblastoma (RB) protein to control exit from the G1 stage of the cell cycle (11). Inactivation of p16 by promoter CGI methylation is one of the most common and earliest epigenetic events in human cancer (12), and is frequently detected in preneoplastic lesions (13-15). Mice provide an apt model in which to study epigenetic dysregulation of p16 in cancer; in a mouse model of chemically-induced lung cancer, p16 methylation is a very early event (16). Moreover, p16 age-associated hypermethylation is observed in several normal human and mouse tissues (17-19). Together, these data suggest that epigenetic silencing of p16 in aging cells facilitates early abnormal clonal expansion, driving tumorigenesis (20). Directly testing this hypothesis, however, requires the ability to specifically target methylation to the p16 promoter CGI.